Fas Associated Death Domain (FADD) is an adaptor protein that is required for signaling by the Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL) receptors. TRAIL receptors are important therapeutic targets in cancer with six TRAIL receptor-targeted drugs in clinical trials at the current time, several others in pre-clinical development and accumulating evidence suggesting that signaling through endogenous TRAIL is important in the mechanism of action of other anti-cancer treatments including DNA damaging agents, anti-metabolites and histone deacetylase inhibitors. The molecular mechanisms by which FADD activates caspases upon TRAIL receptor stimulation are quite well understood, however mechanisms of TRAIL resistance are still poorly understood and this limits our ability to optimally use the TRAIL receptor-targeted drugs. In the previous funding period we analyzed mechanisms of TRAIL resistance and FADD signaling and made the unexpected discovery that TRAIL receptors induce autophagy and that a FADD inhibitor could induce autophagy implying that FADD negatively regulates autophagy. Because autophagy can affect apoptosis responses in tumor cells, we propose that these activities affect the efficiency by which TRAIL receptor signaling activates the apoptosis machinery and thus kills tumor cells. We have also found that autophagy controls the characteristics of dying cells, particularly the release of an immune regulator called HMGB1 and that this also occurs in a FADD-dependent manner in response to TRAIL. Based on these findings, this competitive renewal focuses on three complementary questions: How does FADD regulate autophagy? What effect does autophagy have on TRAIL receptor signaling? And, does manipulation of autophagy provide a way to improve the anti-tumor effect of TRAIL receptor-targeted drugs? To answer these questions we have the following aims. Aim 1. Determine the role of FADD in regulation of autophagy. This aim tests the hypothesis that FADD negatively regulates autophagy by interaction with autophagy regulators. Aim 2. Determine how autophagy affects signaling by TRAIL-R targeted drugs. This aim tests the hypothesis that FADD's ability to inhibit autophagy serves to coordinate competing signals and thus provide fine control over tumor cell death during treatment with TRAIL R-targeted drugs. Aim 3. Test if autophagy manipulation improves the effectiveness of TRAIL-R targeted drugs in vivo. This aim tests the hypothesis that autophagy inhibition will make TRAIL receptor-targeted drugs (lexatumumab, mapatumumab) more effective and uses a unique set of isogenic tumor cells in which we can determine the relative roles of exogenous and endogenous TRAIL receptor stimuli in the anti-tumor response and the role of autophagy in controlling these responses. These studies should provide new insights into FADD and TRAIL receptor signaling, the role of autophagy in determining the response to anti-cancer therapy and provide a basis for improving the use of TRAIL receptor- targeted drugs in treating people with cancer. PUBLIC HEALTH RELEVANCE: In the last few years it has become clear that a hitherto understudied cellular process called autophagy is an important regulator of cancer development and treatment. However there is considerable confusion about what we should try to do to autophagy to improve cancer therapy- in fact it is not clear whether we should try to inhibit autophagy or stimulate it during treatment of cancer. This grant examines signaling by a protein called FADD, which is required for tumor cell killing after activation of TRAIL receptors. This is important because TRAIL receptors are targeted by at least 6 anti-cancer agents and are also important for tumor cell killing by other drugs that work indirectly through TRAIL. In the previous funding period, we made several discoveries; first we found that FADD (and TRAIL) can regulate autophagy. Second, we found that autophagy can modulate the efficiency of tumor cell killing by various drugs including TRAIL. In this proposal we aim to answer the key questions that arose out of the previous work. We will work out how FADD regulates autophagy, how this activity alters signaling by TRAIL receptors and test whether manipulation of these processes alters the effectiveness of treatment by the TRAIL receptor-targeted drugs that are used in people. These studies should provide a way to improve the use of the various anti-cancer agents that target TRAIL receptors which are already in clinical trials and provide a rationale to allow us to manipulate autophagy during cancer treatment.